Analytica Chimica Acta 446 (2001) 107–114

Application of chemometric methods in searching

for illicit Leuckart amphetamine sources
Waldemar Krawczyk a,∗ , Andrzej Parczewski b
a Central Forensic Laboratory of Police, Al. Ujazdowskie 7, 00-583 Warsaw, Poland
b Faculty of Chemistry, Jagiellonian University, Ul. Ingardena 3, 30-060 Cracow, Poland
Received 7 November 2000; accepted 25 June 2001

Abstract
A chromatogram of contaminants of illicit amphetamine is referred to as contamination profile. The profile depends on the
method and conditions of the drug synthesis. Therefore, it allows to link a drug sample sized by the police to the source of
illicit production as well as enables to find out the links between dealers and users. In our study, statistical and chemometric
methods were applied. The areas of 15 more informative chromatographic peaks were selected and used as original variables
describing the drug samples. The selected peaks formed a profile. As a rule, samples submitted to the police laboratory
are classified using correlation coefficient between the corresponding profiles as a measure of similarity. A preliminary
classification and the affiliation of a given profile to a class is verified by means of principal component analysis and cluster
analysis. Different distances between profiles are used including Euclidean and Pearson ones. The data structure is visualized
in two- or three-dimensional spaces of two or three most important principal components. For the purpose of this study, a
total of nearly 1000 drug samples were tested. It has been concluded that amphetamine samples may be attributed to one
source if the Euclidean distance between the corresponding profiles is less than 1, and the Pearson distance is less than
0.04. This approach proved very effective in identification of drug traffic routes and clandestine laboratories by the police.
© 2001 Elsevier Science B.V. All rights reserved.
Keywords: Forensic research; Illicit amphetamine; Impurities profile; Correlation coefficient; Principal component analysis; Cluster analysis

1. Introduction tion of impurities. On the other hand, amphetamine

The synthesis of amphetamine is accompanied by
various side reactions, which result in certain impurity
level of the final product (i.e. amphetamine). Compo-
sition of impurities is characteristic for method and
conditions used for the synthesis. Moreover, it has
been experimentally proven that changes in conditions
of the synthesis significantly influence the composi-
∗ Corresponding author. Fax: +48-22-8497694.
E-mail addresses: clkchem@kgp.waw.pl (W. Krawczyk),
parczews@chemia.uj.edu.pl (A. Parczewski).
samples obtained in a series of repeated syntheses,
carried out according to the same recipe and using the
same batch of substrates, are of similar composition.
A graphic or numerical representation of composi-
tion of impurities in a drug (here amphetamine) is
referred to as impurities profile, and may include, for
instance, a chromatogram. A profile helps to iden-
tify the method of amphetamine synthesis. In the
hereby study, statistical and chemometric methods
[1–5] were applied in order to classify amphetamine
samples and to recognize a tested sample as the one
belonging to a class of samples originating from the

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 2 7 3 - 9
108 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114
Scheme 1. (a) Formamide; (b) hydrochloric acid; (c) diethyl ether, sulfuric acid.
same source, e.g. the same batch produced by an illicit
laboratory.
The Leuckart amphetamine synthesis proceeds ac-
cording to the following Scheme 1 [6–8]. The main
by-products include, among others, the following co-
mpounds: 4-methyl-5-phenylpyrimidine, 4-benzylpyr-
imidine, α,α-dibenzylmethylamine, N,N-di(β-phenyl-
isopropyl)amine, N,N-di(β-phenylisopropyl) forma-
mide. In addition to these, some not reacted sub-
strates and the impurities in the starting material may
be encountered, e.g. formamide, formic acid, BMK,
dibenzyl ketone [7,8].
The profiling of impurities as a tool to identify illicit
drug samples was also used by other authors. Some
examples are contained in the “Proceedings of Interna-
tional Symposium of Forensic Science”, Tokyo, 1993
[9]. Mitsui and Okuyama used principal component
analysis (PCA) in evaluation of data from GC/MS
determination of amphetamine and methamphetamine
in urine. Cluster analysis (CA) using Euclidean dis-
tance was applied in quantitative assay by comparison
of unknown samples with the known ones. Okuyama
and Mitsui used results of GC/MS hemp analysis for
classification purposes. CA was applied using Eu-
clidean distance for 35 samples examined. Marumo
et al. used trace elements (i.e. inorganic) profiles for
characterization of seized methamphetamine samples
(ICP/MS and AAS were applied in trace analysis).
In their evaluation of analytical data and comparison
of samples the authors used simple graphic repre-
sentations. Inoue et al. examined impurities profiles
of methamphetamine seized in Japan by means of
capillary GC. Similarity between samples was tested
by multivariate analysis using Euclidean distance.
Jonson and Stromberg applied the ‘quotient’ method
to the first clustering step and a supervised learning
method (SIMCA) for classification of illicit Leuckart
amphetamine samples on two hierarchical levels, try-
ing to establish batch and source relations [10,11]. By
means of impurity profiling, Perkal, Ng and Pearson
were able to identify methamphetamine samples as be-
ing produced according to one of the three most com-
mon drug synthesis routs encountered in Australia.
A total of 224 samples seized by police were exam-
ined. Three criteria were taken into account including
the Euclidean distance between the profiles and the
canonical variable analysis was applied [12]. The main
ideas used in the hereby study have been presented in
Ph.D. thesis [8] and in a book by Krawczyk [13].
2. Experimental
2.1. Sample preparation
Amphetamine impurities were extracted by 200 ␮l
of n-heptane (containing 35 mg l−1 of diphenylamine
as internal standard) from a 2 ml buffer solution at pH
7.0 containing 200 mg of amphetamine sulfate. In the
extraction step, the pH was checked and adjusted to
pH 7.0 if necessary.
2.2. Gas chromatography
Gas chromatograph HP 5890 series II (Hewlett-
Packard) with flame ionization detector (FID) and au-
tomatic sampler HP 7673 was used with the following
set-up: splitless injection mode (1 min) with 1 ␮l sam-
ples being injected at the injection port temperature
of 250◦ C, 25 m HP-5 column with 0.32 mm inner di-
 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114
ameter and 0.52 ␮m film thickness, oven temperature:
100◦ C for 1 min, a ramp at 12◦ C/min to 240◦ C, where
it was held for 5.5 min, a ramp at 15◦ C/min to final
temperature, 300◦ C for 10 min.
2.3. Data analysis
Data were analyzed by means of special programs
developed at Central Forensic Laboratory of the Police
(for preliminary classification of profiles) and using
STATISTICA StatSoft (PCA, CA, data visualization).
3. Methodology
In general, the procedure of amphetamine profiling
presented in this paper consists of the following steps.
• Gas-chromatographic analysis of amphetamine
samples submitted to the laboratory. Computer
collection of the data (chromatograms).
• Statistical handling of the data: calculation of the
correlation coefficients and distances (similarity
measures) between profiles, application of the PCA
and CA.
• Appropriate attributing the profiles of the tested
samples to the similarity groups (categories, classes,
clusters); each group is presumed to have originated
from the same illicit source.
In practice, profiling is carried out according to the
procedure as follows. Impurities are separated from
a drug (amphetamine), then pre-concentrated and
analyzed by GC. In this way the impurities profile
(chromatogram) of a tested sample is obtained. Out
of several dozen chromatographic peaks only fifteen,
the most characteristic ones, were selected for further
examination. They correspond to organic compounds
(impurities) whose composition depends significantly
on the method and conditions of amphetamine synthe-
sis as well as other consecutive steps of amphetamine
treatment till the final product is obtained. The areas
of 15 selected chromatographic peaks are introduced
to the database, which is built up ‘dynamically’ as
the samples are submitted to the laboratory for the
analysis.
The first step of data handling includes prelimi-
nary grouping of the profiles. In order to achieve this,
correlation coefficients (r) between the profiles are cal-
109
culated. High coefficient values (r > 0.95) indicate
high probability that the corresponding drug samples
originate from the same source and therefore can be
assigned to the same class. A profile may tentatively
be assigned to a class even if 0.8 < r < 0.95, but
only if a visual comparison of entire chromatograms
provides for a high similarity between them.
Following the procedure described above, nearly
1000 chromatograms were separated into 37 groups.
In order to verify the correctness of the above prelim-
inary classification and to be able to visualize the data
structure, PCA and CA were applied both to the orig-
inal data set as well as to the pre-selected groups of
profiles.
To sum up, the classification and visualization of
the data were carried out according to the following
scheme.
• Tentative classification according to the correlation
coefficients between the profiles as well as their
visual comparison.
• PCA and CA using all fifteen original variables as
well as the principal components (PCs) which ex-
plain 90% of total variance and a visual presenta-
tion of data structure by means of two or three most
informative PCs.
• Application of CA for verification of the tentative
classification of a profile to a given category.
4. Results and discussion
4.1. Principal component analysis (PCA)
The purpose of the investigation was to examine
and visualize the data structure of the 37 groups,
I–XXXVII, pre-selected according to the correlation
coefficient, using the most important PCs. The appli-
cation of PCA has been preceded by data standard-
ization. The results presented in Table 1 concern 11
groups each containing at least 15 members (profiles).
For each group of profiles the number of eigenvalues
higher than 1 and the percent of total variance ex-
plained by the corresponding PCs are given. Also the
numbers of PCs which explain a given fraction of the
total variance are presented.
In Table 1, it can be noticed that in case
of pre-selected groups of profiles there are 2–5
eigenvalues higher than 1. The corresponding PCs
110 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114

Table 1
The results of PCAa
Group NP EV > 1 Variance (%) Number of PCs which explain total variance

>90% >95% >98%

I 141 5 84.8 7 8 10
IV 177 5 77.8 8 10 11
VII 29 3 81.0 3 5 7
VIII 24 4 78.1 7 9 11
X 37 5 81.1 7 9 10
XVI 69 5 74.2 8 10 12
XIX 25 3 82.7 5 7 9
XXII 45 4 81.2 6 8 9
XXIV 42 5 88.9 6 7 8
XXVIII 16 4 90.0 4 5 7
XXXIII 30 2 86.2 3 5 6
Full base 1000 6 65.9 11 12 13
a Columns contain the following data: 1: symbols of the groups; 2: number of profiles in the group; 3: number of eigenvalues higher

than 1; 4: percent of total variance explained by PCs corresponding to the eigenvalues in column 3; 5: number of PCs which explain a
given fraction (%) of total variance.

explain 74.2–90.0% of total variance. For the entire
database there are six such PCs, which explain 65.9%
of total variance. The results demonstrate that peak
areas in the chromatograms (profiles) are not strongly
correlated. This particularly concerns the database of
1000 profiles. It may be concluded that the objects
(profiles) in the 15-dimensional space of original vari-
ables (peaks areas) are not set up regularly and the
arrangement vary considerably from group to group.
A common problem appears in connection to the
criterion for selection of the number of PCs necessary
to proper describing the original data structure in a re-
duced space. The authors decided to use the percent-
age of total variance explained as the criterion (see last
columns in Table 1). The examples of dependence of
the cumulative variance explained versus the number
of PCs have been presented in Fig. 1a and b.
An advantage of the PCA application is the possi-
bility of at least partial visualization of data structure
(distribution of objects in the 15-dimensional space)
in two- or three-dimensional space of the most infor-
mative PCs. A projection of objects onto this reduced
space remains the most characteristic features of the
original structure, e.g. concerning clusters and out-
liers. An example is presented in Fig. 2. The projec-
tion presented in Fig. 2 well reflects the arrangement
of objects in the original 15-dimensional space. It can
be seen that there are samples which differ signifi-
cantly from the distinct root of the cluster. They seem
to be outliers, i.e. they may not belong to the same
source as do the samples making the root of the clus-
ter. Their membership to the class has to be carefully
checked.
4.2. Cluster analysis (CA)
The effectiveness of the preliminary classification
carried out on the basis of the correlation coefficient
is also verified by means of CA. The decision has to
be undertaken concerning a choice of the appropriate
distance or similarity measure between the profiles
and a clustering method. Another factor which may
influence the results of CA is pre-treatment of data
(e.g. standardization).
Different distances are used in CA, e.g. Minkowski
including Manhattan and Euclidean, square Euclidean,
Chebyshev, Pearson based on the correlation coeffi-
cient and Mahalanobis. Which distance is appropriate
depends on context and purpose of data analysis. Var-
ious distances were dealt with in our investigations. In
the examples below we used Euclidean and Pearson
distances.
There are various approaches in CA used in form-
ing the clusters, e.g. in the hierarchical agglomerative
clustering, the most common one, several methods are
applied including single linkage (nearest neighbor),
 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114 111

Fig. 1. Cumulative variance explained vs. number of PCs: (a) for the group IV (177 profiles) and (b) for entire database (ca. 1000 profiles).

complete linkage (furthest neighbor), average linkage
with weighted or unweighted pair group methods. In
our study we used the single linkage method. The re-
sults of CA are usually presented graphically in form
of a dendrogram (Fig. 3). Along the horizontal line
(abscissa) the symbols of objects are shown and along
the vertical axis (ordinate) the distance between the
objects is given.
Fig. 3 presents dendrogram with pre-selected
groups XXII and XXIV (Table 1) generated by means
of Euclidean distances. The subgroups are observed,
where the distances between the objects are very small
and which correspond to very similar drug samples.
In such subgroups the Euclidean distance is less than
0.3 (Pearson distance less than 0.01, Manhattan—
less than 0.5 and Chebyshev—less than 0.1). It may
112 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114
Fig. 2. Score plot. The projection of profiles in groups XXII and
XXIV (see Table 1) on to PC1, PC2, PC3 space (factor 1 vs.
factor 2 vs. factor 3 space).
Fig. 3. Dendrograms for the pre-selected groups of profiles XXII and XXIV. Euclidean distances applied.
be assumed that the subgroups involve amphetamine
samples, which originate from the same production
batch.
The main purpose of application of CA to the
proposed analysis scheme is to confirm preliminary
classification of the profiles made on the basis of corre-
lation coefficients. The dendrogram obtained with Eu-
clidean distance is particularly useful in checking new
samples (profiles) which were assigned to a group with
rather low correlation coefficient (0.8 < r < 0.95).
A careful inspection of many real-case CA diagrams
and taking into the account knowledge and practice in
the field of drug characterization and gas chromatog-
raphy, leads to a conclusion that the profiles can be
considered similar (i.e. amphetamine samples origi-
nate from the same source) if the Euclidean distance
appears less than 1, and the Pearson distance is less
than 0.04.
The above thesis is also supported by a model exam-
ple based on 50 amphetamine profiles taken from five
most numerous pre-selected groups: I, IV, XVI, XXII
and XXIV (see Table 1). From each group 10 profiles
 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114
Fig. 4. Dendrograms for the model data base composed of the selected members of five pre-selected groups of profiles (see text). Euclidean
distances applied.
were randomly selected out of those for which the cor-
relation coefficients exceeded 0.95 within a subgroup.
In this way, a model database with 50 profiles, each
described by 15 peak areas was generated. The results
of CA have been presented in Fig. 4. Dendrogram re-
veal five distinct groups (clusters). The Euclidean dis-
tance between the clusters exceeds 1 and the Pearson
distance exceeds 0.04. Actually, the profiles attributed
to a group in Fig. 4 belong to the same illicit source.
In Fig. 2 score plot and Fig. 3 dendrogram are
presented which concern two pre-selected groups of
samples, XXII and XXIV (see Table 1). All mem-
bers of each group were taken into account including
profiles for which 0.8 < r < 0.95 as compared with
those forming a close core of a group. Fig. 2 reveals
two distinct clusters with some samples that clearly
differ from the clusters’ cores (see cases in Fig. 3 for
which Euclidean distance exceeds 1). These samples
require special examination including comparison of
entire chromatograms. Data presented in Figs. 2 and 3
prove credibility and effectiveness of the pre-selection
of amphetamine samples according to the correlation
coefficient. On the other hand, the application of PCA
and CA makes visualization of data structure possible
using different similarity/dissimilarity measures, e.g.
113
Euclidean distance used in the examples presented
above. The examples presented above in Figs. 2–4
show that Euclidean distance appears useful not only
in indication of outliers inside a pre-selected group of
samples (profiles) but also in discrimination between
different groups.
5. Final remarks
Chemometrics becomes a more and more useful
tool in searching for clandestine laboratories and drug
traffic. Profiling of impurities produces data that are
handling according to the statistical and chemometric
methods such as PCA and CA. They are used in or-
der to classify drug samples to appropriate similarity
groups.
Efficiency of the approach presented in a hereby
study has been proven on more than 1000 real drug
samples confiscated by the police. The method enables
an objective computer aided comparison and classifi-
cation of amphetamine profiles. On the other hand, the
approach includes an interactive step, at the beginning
of the classification procedure, in which the whole
chromatograms may be compared and the appropriate
114 W. Krawczyk, A. Parczewski / Analytica Chimica Acta 446 (2001) 107–114
decision can be undertaken concerning assignment of
a sample (profile) to the pre-selected groups. The ap-
proach proved very helpful in the operations of the
police against illicit amphetamine manufacturers and
dealers in Poland and therefore the authors will con-
tinuously work on improving efficiency of the method.
A very characteristic point of the described appli-
cation of chemometric methods is that it concerns a
‘dynamic’, evolving system under consideration. This
means that as drug samples seized by the police are de-
livered to the lab their profiles are handled according
the proposed approach, and the corresponding points
in the multi-component space of features are added to
the previous patterns. Actually, the patterns expand. As
long as a batch of illicit drug production is still on the
illegal market, and the drug samples are still seized by
the police, the population of the corresponding clus-
ter increases. A new drug source results in the forma-
tion of a new cluster, while if the drug stock of an old
production batch is running low no new profiles are
added to the existing cluster which becomes stagnant.
Then the examples presented in this paper corre-
spond to a given moment in time and the patterns be-
fore and after that time differ their changes reflecting
the change in drug marked and distribution. Of course,
for the police operation purposes each profile is de-
scribed by the time, place and other conditions and
circumstances the drug sample was seized.
Although capillary GC/MS turned out to be useful
in amphetamine profiling, some additional significant
(informative) variables, apart from the areas of chro-
matographic peaks, will be taken into consideration,
e.g. inorganic impurities profile. The usefulness of var-
ious pattern recognition methods is already tested.
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